GSM6932279: Prostate tip luminal cells 2; Mus musculus; RNA-Seq
Minced tissues were digested in 5mg/ml Collagenase Type I (Sigma-Aldrich, diluted in HBSS) for 2 h at 37 °C under agitation.  Cells were rinsed in HBSS supplemented with 10% FBS and the cell suspensions were filtered through a 70um cell strainers (BD Bioscience), followed by 2 successive washes in HBSS supplemented with 2% FBS. Samples were incubated in 200 μl PBS supplemented with 2% FBS with fluorochrome-conjugated antibodies for 30 min on ice protected from light. Antibodies were washed with 2% FBS/PBS and cells were resuspended in 2.5 mg/ml DAPI (Invitrogen, D1306) before analysis. The following antibodies were used: PE-conjugated anti-CD45 (rat, clone 30-F11, dilution 1:100, BD Biosciences Cat#553081), PE-conjugated anti-CD31 (rat, clone MEC 13.3, dilution 1:100, BD Biosciences Cat#553373), PE-conjugated anti-CD140a (rat, clone APA5, dilution 1:100, BD Biosciences Cat#624049), APC-conjugated anti-CD49f (rat, clone GoH3, dilution 1:100, eBioscience Cat#17-0495), APC-Cy7-conjugated anti-EpCAM (rat, clone G8.8, dilution 1:100, BioLegend Cat#118218). Living cells were selected by forward and side scatter, doublets discriminating and DAPI dye exclusion. CD45+, CD31+ and CD140a+ cells were excluded (Lin+) before analysis of the epithelial basal (EpCamhigh/CD49fhigh) and luminal (EpCamhigh/CD49flow) cells. Fluorescence-activated cell sorting and analysis were performed using FACSAria and LSRFortessa, using FACSDiva software (BD Bioscience). Sorted cells were collected in lysis buffer for RNA extraction (RLT buffer, QIAGEN). RNA extraction from FACS isolated cells was performed using RNeasy micro kit (QIAGEN) according to the manufacturer´s recommendations. Prior to sequencing the quality of RNA was evaluated by Bioanalyzer 2100 (Agilent). Indexed cDNA libraries were obtained using the Ovation Solo RNA-seq Systems (NuGen) following manufacturer's recommendations.